1. Invention Field
The present invention teaches various designs for bioreactors, including an RBC (rotary biological contactor) wherein there is provided, in the preferred embodiment of the present invention, a plurality of barrier members enveloped in a chamber, configured to provide a plurality of nutrient chambers for microorganisms or cells.
The preferred embodiment teaches a plurality of disk configured, rotating chamber partition members, enveloped by a generally horizontally situated, cylindrical chamber member, and wherein there may be further included a system to vary the nutrient environment of said micro-organisms or cells, including means to individually vary the temperature and/or atmosphere of each of said chamber areas.
Alternative embodiments of the present invention teach the utilization of various permeable cellular support media in facilitating an optimal system for retaining and nurturing various micro-organisms or cells, coupled with a rotating or rolling RBC or Bioreactor.
2. General Background Discussion
Several types of cells, especially fungi, plant and mammalian cells, require oxygen for growth and sustenance. Many antibiotics, including such organisms as penicillin and streptomycin, also require oxygen. In addition, various strains of living tissues and organisms have been found to metabolize and break down various toxic and/or heretofore environmentally undisposable materials.
For example, the fungus Phanerochaete chrysosporium (white rot) has been found to degrade TNT (trinitrotoluene) as well as chlorine compounds in pulp mill effluent. The fungus Rhizopus arrhizus is able to leach out toxic metals, while Rhizopus oryzae makes lactic acid for biodegradable polymers. The fungus Aspergillus niger is able to synthesize citric acid and food flavors. Mammalian cells have been known to produce interferon. Plant cells produce alkaloids.
The advantages of plug flow are well known to engineers, in accelerating the metabolism of these various materials by the living tissues, due in part to plug flows suitability for providing continuous reactions. With plug flow, product inhibition is low in the first section, but this is not true when back mixing takes place. In an effort to provide plug flow in an RBC (rotary biological contactor) with solid plastic discs, baffles are employed, but these are not very effective because liquid can go around the baffle and by-pass the discs. The prior art has yet to contemplate an RBC configured to provide better plug flow. Even further, the prior art has not contemplated a bioreactor which may be utilized in conjunction with a variety of permeable cell supports or various culture media, prior art designs being largely limited to specific applications with little diversity.
While the generalized concept of the RBC has been well contemplated in the prior art, what is needed is an improved reactor wherein the operating efficiency and growth viability, and versatility, is substantially enhanced.
U.S. Pat. No. 4,655,926 by Chang et al contemplates a rectangular tank with rotating solid plastic discs for retaining the cell cultures in several chamber sections, further teaching oxygen fed into one end of the tank, run in a batchwise fashion. This is as opposed to a continuous flow system, which requires less labor and is more efficient, avoiding product inhibition near the end of the run. When running continuously, feeding oxygen on one end, product inhibition is low in the first section. Further, with solid plastic discs, cells grow only on the outside of the disc. On the other hand, if the discs were comprised of a permeable material, such as polyurethane foam, cells would grow on the inside of the disc also.
Instead of having straight sides of the tank, when the tank is curved, just slightly larger diameter than the discs and run half full, liquid and oxygen feed is forced through the permeable discs and better contact is made and no foaming is experienced. Accordingly, liquid permeates the bottom half of the discs, while the gas permeates the top portion.
With the prior art solid plastic discs, cells are found only on the outside of the disc, limiting the habitable area by two dimensions. This is as opposed to a three dimensional disc system, wherein significantly more cells could be employed. Solid plastic, like stainless, is hard, completely impermeable to fluid medium, and is not conducive to cell growth, as opposed to a "soft" surface.
A list of prior patents which may be of interest is presented below:
______________________________________ U.S. Pat. No. Patentee(s) Issue Date ______________________________________ 3,594,277 Mako 07/20/1971 4,468,326 Kawert 08/28/1984 4,554,075 Chang et al 11/19/1985 4,655,926 Chang et al 03/07/1987 4,877,731 Ling et al 10/31/1989 4,963,486 Hang 10/16/1990 4,999,302 Kahler et al 03/12/1991 ______________________________________
Publications which may be of interest may include:
Miller, W., Wilke, C., and Blanch, H.: Effects of Dissolved Oxygen Concentration on Hybridoma Growth and Metabolism in Continuous Culture. Jour Cellular Phys 132:524-530 (1987).
Webb, C. and Mavituna, F.: Immobilization of Plant Cells in a Reticulated Foam Matrix. Plant and Animal Cells, Holstead Press - J. Wiley (New York) 94-99.
Oller, A., Buser, C., Tyo, M., and Thilly, W.: Growth of mammalian cells at high oxygen concentrations. Jour Cell Sci 94, 43-49 (1989).
Facchini, P., and DiCosmo, F.: Plant Cell Bioreactor for the Production of Protoberberine Alkaloids from Immobilized Thalictrum rugosum Cultures. Biotech and Bioeng 37, 397-403 (1991).
Chiou, T., Murakami, S., Wang, D., and Wu, W.: A Fiber-Bed Bioreactor for Anchorage-Dependent Animal Cell Cultures: Part I. Bioreactor Design and Operations. Biotech and Bioeng 37, 755-761 (1991).
Sakurai, A. and Hiroshi, I.: Effect of Operational Conditions on the Rate of Citric Acid Production by Rotating Disc Contactor Using Aspergillus niger. Jour Ferm and Bioeng. Vol 73, 3, 251-254 (1992).
Tiwaree, R., Cho, K., Hirai, M. and Shoda, M.: Biological Deodorization of Dimethyl Sulfide Using Different Fabrics as the Carriers of the Microorganisms. Applied Biochem and Biotech 32, 135-148 (1992).
NSW Corporation: Biofilter Media, RBCs and Tower Packings. Sales Brochure.
Heidman, J. A., Brenner, R. C., and Gilbert, W. G.: Summary of Design Information On Rotating Biological Contactors. EPA-430/9-84-008.
U.S. Pat. No. 4,554,075 to Chang is similar to '926 above. In col 3, line 9, it is stated that said system can operate in a plug flow mode, however, "plug flow" by definition means no back mixing will occur, and there is back mixing in the taught apparatus, in the first compartment, when liquid enters the first compartment it can by-pass the first disc and go to the second disc.
U.S. Pat. No. 4,468,326 to Kawert teaches a system (col 2 line 19) wherein he says baffles are arranged so as to prevent water from shunting past the rotors. However, with thin discs, media could by pass discs when baffles (7) are far apart as in FIG. 1b. Curved tank sides close to the discs would prevent by-pass. When the sides of the tank are curved and close to the disc, baffles are not required.
Iyer gave talk 100 BIOT at the American Chemical Society Meeting in San Francisco in April, 1992, wherein he disclosed his ability to produce lactic acid with the fungus Rhizopus oryzae or arrhizus, utilizing an RBC having solid plastic discs.
Hang, in U.S. Pat. No. 4,963,486 also has a process for making lactic acid with this fungus. Lactic acid polymers are biodegradable, so this should reduce landfills, which are piling up. Ling, in U.S. Pat. No. 4,877,731, describes such a process.
Conventional methods of adding oxygen involve bubbling the gas up from the bottom of the RBC, through the liquid medium. However, the solubility of the gas in water is only about 10 ppm. There is a stagnant region around the bubble, then it has to go through the bulk liquid, another stagnant region around the cell and then through the cell wall for the oxygen to get to the cell. This is shown in the publication Biochem Eng Fundamentals (2nd ed), Baily and Ollis (McGraw Hill) p. 460.
Immobilization of the plant cells in reticulated foam depends upon the size of the cell, as far as picking the size of the pores. In the publication Plant and Animal Cells, Webb and Mavituna (John Wiley) Ch 6 illustrates the depth of entrapment in 10 pores per inch, 30 ppi and 40 ppi (pg 97). They also describe a method of getting smaller cells by homogenization (p 98). It would be desirable to have larger pore sizes near the entrance of the reactor, and smaller pores near the exit, in order to entrap the largest number of cells, while allowing for gas permeability. One such method, uncontemplated in the prior art, could entail the combining of foam and rice hulls.
Rice hulls contain cellulose, hemicellulose, silica (about 20-23%) and other metals needed for growing Phanerochaete chrysosporium. Soybean hulls contain about 8% protein. Cellulose has adjacent glucose fragments linked in a 1,4 beta linkage, whereas starches are 1,4 alpha linkage and are easier to break down, but Phanerochaete chrysosporium has cellulase enzymes, so if a little water is added, it can break down cellulose, but if the hulls are coated with a little starch-glucose, it reacts faster.
Instead of oxygen, odor causing gasses such as dimethyl sulfide and hydrogen sulfide can be removed with organisms on fabrics, as described in the above referenced article entitled "Biological deodorization of dimethyl sulfide . . . " in Applied Biochem and Biotech. These are the very gasses emitted by paper mills.
Formaldehyde gas is found in morgues and factories using urea formaldehyde resins. It is soluble in water, and Professor Moyer of the Biology Department at Trinity University has found an organism, which he has grown on polyester fiber, that degrades it.
Plant and mammalian cells also require oxygen. Many of these cells are fragile (sheer sensitive) and when oxygen is bubbled up from the bottom of a conventional fermenter, large air compressors are needed and the bubbles burst at the surface, causing foam and damage to the cells. To avoid this, Wang et al, in Biotech and Bioeng 37, 755-76 with a vertical reactor (not an RBC), put air up the lower middle portion of the containment vessel, with the liquid configured to overflow around the outside of the partitions, flowing down through fiberglass. M. Lavery, and A. W. Nienow infused silicone antifoam (6 ppm) to reduce the K.sub.L a value by ca.50% in their paper Oxygen Transfer in Animal Cell Culture Medium Biotech and Bioeng, Vol XXX, P 368-373 (1987). CHO (chinese hamster ovary) cells produce interferon. Wang says that this design could be scaled up (p 756), but their unit was only 35 cm high, and if it were much higher, the oxygen would be depleted as it reached the bottom.
Wang further asserts (p 760) that the "top layers had more cells than the bottom layers". Chang ('926 reference) asserts that oxygen concentration is important (col 4 line 37). Facchini says (p 402) more cells near the perimeter.
In the design of the present, applied for invention, oxygen distribution is configured such that concentration is uniform throughout the various partitions, with almost the same concentration from end to end, but if the support causes some pressure drop, more oxygen can be put in the top of the middle section 103, as shown in FIG. 1. As solubility of the gas in water is relatively very low, direct contact is better.
When the gas is pressurized in the headspace above the liquid, even better solubility is obtained. Plant cells also produce valuable alkaloids. Facchini and DiCosmo, Biot and Bioeng 37, 397-403 produced alkaloids on glass fiber, but it took several days. With a more efficient reactor, the time could be shortened. There were more cells near the perimeter (pg 402) since air reached them better.
Heidmann, Brenner, and Gilbert summarize the state of the art in RBC's in their treatise Summary of Design Information on Rotating Biological Contactors (EPA 1984):
"Each manufacturer designs its own shape, size, and thickness of shaft, the wall thickness of the shaft is governed by structural requirements, and the shape is highly dependent on the method the manufacturer employ in supporting the plastic media from the shaft. The five manufacturers each utilize a shaft that differs from the others in either the thickness, size, or shape, or in some cases all three. Structurally, these differences are readily apparent as shown in FIG. 2 and identified in Table One. PA0 MEDIA PA0 The heart of the RBC process is the plastic media. In 1972, the high density polyethylene (HDPE) disc was introduced as a cost reduction alternative the previously used 0.5 inch thick polystyrene disc. The major advantage of polyethylene is its ability to be formed into various configurations that require a thickness of only 40 to 60 mils (0.04 to 0.06 in.). This innovation enabled 100,000 to 180,000 square feet of surface area to be provided on a 27 foot shaft with 12 foot diameter media. Today, all U.S. manufacturers of RBC's utilize polyethylene as their plastic media. PA0 SECTION 2 PA0 PROCESS DESCRIPTION PA0 All RBC systems are cylindrical-type structures consisting of plastic media attached to and/or supported by horizontal rotating shafts. The first commercial RBC system was installed in West Germany in 1960. Units constructed from this time to the early 1970's used flat 0.5 inch thick, 6.5-10 foot diameter expanded polystyrene disks. All present systems use thin (0.04 to 0.06 in) high density plastic media either formed as discs or sections of discs and aligned perpendicular to the shaft, or spirally wound onto and aligned parallel to the shaft."
Thus, the prior art RBC's have been of largely limited structure and usage, its limitations to large extent brought about due to the relatively impermiable, two dimensional solid cell supports, as opposed to a three dimensional, fluid and cell permiable support, as is contemplated in the present, applied for invention.